1. Field of the Invention
This invention relates generally to uses of an animal model for repetitive behaviors, including symptoms of obsessive compulsive disorder and related behavioral disorders.
2. Background
Hoxb8 is a member of the mammalian Hox complex, a group of genes whose origins have been traced prior to the evolution of animal species. The Hox complex, or Homeobox-containing gene complex, comprises a group of 39 genes best known for their role in cuing positional information during embryogenesis. Like most Hox genes, Hoxb8 is first expressed at approximately E7.5 in the posterior region of developing embryos. During the next twenty-four hours, Hoxb8 expression continues to move anteriorly so that by E8.5 this gene is expressed to the level of somite 5 in the neuroectodenn and to the somite 10/11 boundary in the mesoderm. Later in development (E11.5), Hoxb8 is expressed in each of the developing spinal ganglia and throughout the spinal cord to the level of the hindbrain/spinal cord boundary (Deschamps and Wijgerde, 1993). Further studies have demonstrated that Hoxb8 expression in the spinal cord is restricted to portions of the dorsal and ventral halves of the developing cord (Graham et. al, 1991; van den Aklcer et. al., 1999). Within the prevertebra (PV), PV8 is the most anterior PV to express high levels of Hoxb8, although weak expression has been demonstrated in PV7. Additionally, Hoxb8 expression has been described in the zone of polarizing activity (ZPA) in the developing forelimb bud.
The colinear arrangement of genes within the Hox clusters of metazoan organisms has led to the speculation that this organization is an evolutionarily constrained requirement of Hox gene function. The sequential activation and maintenance of Hox gene expression in an ordered fashion that reflects the chromosomal position of each gene along the A-P axis of metazoan embryos, is determined through the concerted action of cis-acting regulatory sequences that govern expression of each gene (Gerhart and Kirschner, 1997). Since the protein-coding regions of Hox genes are often quite small, the vast majority of the sequences contained within Hox clusters are occupied by potential regulatory elements. These cis-regulatory networks appear to be responsible for the initial expression of Hox genes in a colinear fashion that reflects the order of genes on the chromosome, however, the precise mechanisms of Hox gene activation are not completely understood. The initial activation of Hox gene expression as a colinear single unit would provide an evolutionary advantage over systems requiring independent action to activate each individual gene, since fewer signals would be required for activation of the colinear network.
The Hox clusters have also acquired properties that are responsible for the maintenance of Hox protein expression networks along the A-P axis of metazoan embryos. Following initial activation of Hox gene expression, the continued expression of Hox genes throughout subsequent developmental stages is maintained by the Hox proteins themselves, through a variety of interactions with the cis-acting regulatory sequences within the cluster.
In developing metazoans, Hox gene expression begins in the posterior end of the embryo and then spreads anteriorly to reach sharp borders of expression along the A-P axis of embryos. As a consequence, the expression of many Hox genes is seen along the entire A-P axis posterior to their individual anterior limits of expression. This leads to overlapping expression of many Hox genes in posterior compartments. In order to limit the action of an individual Hox protein within the anterior-most compartment that it is expressed in, some Hox proteins have developed a hierarchy of repressive activity known as posterior prevalence or posterior dominance (Gonzalez-Reyes and Morata, 1990; Morata, 1993). In this hierarchy, a Hox protein that is expressed in a posterior compartment represses the expression of anterior genes within that same compartment. This property results in the sharpening of compartment boundaries in developing embryos, and even more importantly, is a consequence of Hox protein action within the Hox cluster. A second interesting feature that has been associated with the maintenance of Hox protein expression is autoactivation. Individual Hox proteins have been demonstrated to maintain their own expression within an individual segment following initial activation, by binding to and activating their own enhancers. For example, in mice autoregulation of the Hoxb1 locus has been demonstrated (Gavalas et al., 1998; Goddard et al., 1996; Maconochie et al., 1997; Ogura and Evans, 1995a; Ogura and Evans, 1995b; Studer et al., 1998; Yan et al., 1998).
Interestingly, after the initial establishment of compartment boundaries, the regionalization of cell types within individual compartments is independent of what is happening in other compartments. This is exemplified by the phenotypes associated by loss of Hox gene function. When an individual Hox gene is inactivated, in many instances the observed phenotypes are characterized by transformations to the identity of a neighboring compartment. However, even though the identity of a compartment may be altered, further activation and function of the rest of the Hox cluster is unaffected. As a consequence, in most instances, organisms mutant for an individual gene often display phenotypes associated with the compartment the gene is normally expressed in, however development of the rest of the embryo proceeds unaltered (Gerhart and Kirschner, 1997).
This compartmentalization of embryos following Hox gene activation has profound consequences with respect to the evolution of body patterns in metazoan organisms. Once the identity of a compartment has been determined through activation of the Hox network, the action of an individual Hox protein within a segment is determined by the interaction of the protein with the cis-regulatory sequences of the target gene. Throughout evolution, mutation and selection in the cis-acting regulatory sequences of potential target genes would have determined the regulatory interaction between a particular Hox protein and the target gene. As a consequence, interactions with different types of target genes within an individual segment may result, leading to changes in the both the appearance and function of that segment in developmentally mature metazoans. Thus, the varied body patterns that result from the identical segmental location and expression of orthologous Hox proteins in different metazoa may be the result of the evolutionary conservation of a flexible developmental program.
During vertebrate evolution, quadruplication of the Hox cluster resulted in an increase in both the amount of Hox protein present in an individual compartment, as well as the number of cis-acting regulatory sequences controlling Hox gene expression. Paralogous Hox proteins that were expressed in the same compartment may have been able to activate and repress one another, since they probably shared common regulatory sequences. Consequently, vertebrate paralogous Hox genes have diverged in two ways: through changes in the cis-acting regulatory sequences and through changes in the protein coding sequences. Changes in the cis-acting regulatory sequences may have produced changes in the amount of a particular protein present or altered the location or timing of expression for the individual gene. Changes in the protein-coding sequences could have resulted in alterations in the specificity of the homeodomain for DNA, as well as alterations in interactions with other proteins. As a result of changes in both the cis-acting regulatory sequences and protein coding sequences, a single member of a paralogous group could evolve interactions with its own set of target genes, thereby producing novel morphologies within a compartment.
Previous analyses of both the expression pattern and function of Hoxb8 had suggested that this gene was crucial to the development of forelimb buds (Deschamps and Wijgerde, 1993). In the developing forelimb bud, the posterior margin, the zone of polarizing activity (ZPA), is characterized by expression of Sonic hedgehog (Shh) and the 5′ Hoxd genes (Dolle et al., 1989; Nelson et al., 1996). Transplantation of either the ZPA or Shih-secreting cells to the anterior margin of the limb bud results in mirror-image expression patterns of the 5′ Hoxd genes, as well as mirror-image digit patterns (Izpisua-Belmonte et al., 1991; Nohno et al., 1991; Riddle et al., 1995). Expression of Hoxb8 in the developing forelimb is restricted to the posterior half of the structure. Ectopic expression of Hoxb8 using the retinoic acid receptor-s promoter to drive expression in regions anterior to the normal limit of Hoxb8 expression resulted in the expression of Shh in the anterior portion of the limb bud (Charite et al., 1994). Furthermore, these Hoxb8 transgenic mice also developed a second ZPA in the anterior portion of the forelimb bud that was characterized by expression of the 5′ Hoxd genes, resulting in the formation of mirror-image duplications of the forelimbs. These experiments suggested that Hoxb8 played a crucial role in the establishment of the ZPA and thus the patterning of the posterior half of the forelimb bud.
Although expression of Hox genes other than Hoxb8 in adult tissues, including the CNS, have been described, most studies assessing Hox protein function have been limited to embryological studies. Consequently, whether the activity of Hox proteins in adult tissues results from conserved interactions or through novel interactions is unknown. Previous analyses of the cis-regulatory elements surrounding the Hoxb8 gene have indicated that expression of this gene is governed by the concerted action of numerous enhancer elements (Charite et al., 1995; Valarche et al., 1997; Zwardtlis et al., 1992). Prior to the present invention, a role for the expression product of Hoxb8 in the central nervous system or in complex behaviors such as grooming behaviors have never been elucidated.
Obsessive-compulsive disorder (OCD) is a condition that is characterized by obsessions such as fear of contamination and/or such repetitive behaviors as excessive cleanliness. Epidemiological studies using cross-national representations have indicated that this disorder is quite common, with a prevalence rate ranging from 1.9-2.5 per 100 in seven different international communities (Horwath and Weissman, 2000). A genetic component for this disorder is suggested by twin studies, where concordance rates as high as 87% between monozygotic twins have been reported. Additionally, family studies have indicated rates of OCD as high as 10.9% in first-degree relatives of OCD probands (Wolff et al., 2000).
Repetitive behaviors are also associated with other disorders such as Tourette syndrome, which is characterized by repetitive tics and utterances, and trichotillomania, which is characterized by the removal of one's own body hair. The national Tourette Syndrome Association, Inc. used to publish estimates suggesting that Tourette syndrome affected only 1 in every 10,000 people. More recent evidence, however, evidence suggests that 2 to 3 out of every 100 children or teenagers may have some form of this spectrum disorder. In the case of trichotillomania, there have been no epidemiological studies to identify the actual number of people with this condition yet. It is estimated that in the United States alone, there are probably between 6 to 8 million sufferers of trichotillomania.
Despite the prevalence of repetitive behaviors, few animal models exist to facilitate the study of the underlying basis for or treatments of such behaviors. Proposed animal models include dogs with canine acral lick dermatitis (Rapoport, et al., 1992), a condition in which the animal licks its paws or flank to a point that ulcers and infection develop, or other animals that show displacement behaviors, such as grooming, upon stress. (Moon-Fanelli et al., 1999; Fentress, 1988; Sachs, 1988) Finally, normal behaviors such as marble burying in wild-type mice have been used as models for repetitive behaviors. (Londei et al. 1998; Ichimaru, 1995; Njung'e and Handley, 1991) Prior to the present invention, however, no genetic model for repetitive behavior.